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received: 11 December 2015 accepted: 21 April 2016 Published: 10 May 2016
Efficient Photocatalytic Disinfection of Escherichia coli O157:H7 using C70-TiO2 Hybrid under Visible Light Irradiation Kai Ouyang1,*, Ke Dai1,*, Sharon L. Walker2, Qiaoyun Huang1, Xixiang Yin3 & Peng Cai1 Efficient photocatalytic disinfection of Escherichia coli O157:H7 was achieved by using a C70 modified TiO2 (C70-TiO2) hybrid as a photocatalyst under visible light (λ > 420 nm) irradiation. Disinfection experiments showed that 73% of E. coli O157:H7 died within 2 h with a disinfection rate constant of k = 0.01 min−1, which is three times that measured for TiO2. The mechanism of cell death was investigated by using several scavengers combined with a partition system. The results revealed that diffusing hydroxyl radicals play an important role in the photocatalytically initiated bacterial death, and direct contact between C70-TiO2 hybrid and bacteria is not indispensable in the photocatalytic disinfection process. Extracellular polymeric substances (EPS) of bacteria have little effect on the disinfection efficiency. Analyses of the inhibitory effect of C70-TiO2 thin films on E. coli O157:H7 showed a decrease of the bacterial concentration from 3 × 108 to 38 cfu mL−1 in the solution with C70-TiO2 thin film in the first 2 h of irradiation and a complete inhibition of the growth of E. coli O157:H7 in the later 24 h irradiation. Millions of people die from diseases transmitted through contaminated water or human excreta every year1. As a waterborne pathogenic microorganism, Escherichia coli O157:H7 reportedly causes an estimated 73,480 illnesses, 2,168 hospitalizations, and 61 deaths each year in the United States2. Reliable, efficient, and environmentally safe disinfection has become an increasing concern throughout the world3. Traditional water disinfection methods such as chemical oxidation technologies can require expensive chemicals or costly equipment. Moreover, unwanted oxidation bi-products may be formed in water from chlorination or ozonation processes4. Although UV radiation is capable of sustained disinfection, the hazards of intensive and direct use of UV radiation limit its application5. Therefore, the development of an efficient and environmental friendly disinfection technology has become an urgent demand. Since Matsunaga et al. discovered the bactericidal activity of TiO2 as a photocatalyst for the first time in 19856, many studies related to the bactericidal effect of TiO2 photocatalyst have been reported, including the death of bacteria and inactivation of viruses7,8. However, the potential for the practical application of TiO2 is restricted by the broad band gap (3.2 eV) of anatase TiO2 and low quantum efficiency9. Recently, considerable effort has been taken to address these issues by doping the TiO2 with non-metal or metal elements or by coupling with narrow band semiconductors10–12. Carbon nanomaterials (fullerene, carbon nanotube, and graphene) are highly valued because of their excellent optical performance, small size effect, and ease in achieving surface functionalization towards coupling with TiO213. C60 has drawn particular attention of researchers due to its being an excellent electron acceptor with delocalized conjugated structure14–16, which can efficiently separate photo-induced charge. C70 is a close-shell configuration consisting of 35 bonding molecular orbitals with 70 p-electrons, which can promote efficient electron transfer reduction17. Compared with C60, C70 has higher electron affinity and higher possibility to form anions or free radicals because of the reduced symmetry structure18. In addition, C70 has a larger photo cross-sectional area, which suggests that a high light harvesting efficiency can be achieved19. Latest 1 State Key Laboratory of Agricultural Microbiology, College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China. 2Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521, USA. 3Jinan Research Academy of Environmental Sciences, Jinan 250014, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to P.C. (email: [email protected])
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Figure 1. Survival of E. coli O157:H7 (0.5 mg mL−1) presented as the ratio of live to total cells (Nt/N0) versus the time. Suspensions of cells were exposed to dispersions containing different catalysts (1 mg mL−1) in the dark and under visible light irradiation.
studies revealed that C70-TiO2 hybrids show a significant photocatalytic activity for degradation of organic pollutant in visible light region20,21. The aforementioned reports imply that a high efficient bactericidal activity can be expected from the C70-TiO2 hybrid. In the present study, we prepared a C70-TiO2 hybrid by a hydrothermal reaction process and investigated this material’s capacity for photocatalytic disinfection of E. coli O157:H7 under visible light irradiation. The effects of the photocatalyst dosages and the contact modes between C70-TiO2 hybrid and bacterial cells on the photocatalytic disinfection rates were investigated. Several scavengers for hydroxyl radicals and valence-band holes were applied to examine the roles of the oxidative species in the photocatalytic disinfection of E. coli O157:H7. Moreover, the effect of bacterial extracellular polymeric substance (EPS) on the photocatalytic disinfection was also evaluated.
Results and Discussion
Crystal Phase Composition, Morphology and Optical Properties of C70-TiO2 Hybrid. The XRD
patterns of TiO2 and C70-TiO2 hybrid are shown in Supplementary Fig. S1. The peaks at 25.4° (101), 37.5° (004), 48.1° (200), 53.8° (105), 54.8° (211), and 62.8° (204) corresponded to the pure anatase phase. It can also be seen that the diffraction peaks of anatase phase in C70-TiO2 hybrid did not shift with the incorporation of C70, indicating that the introduction of C70 does not affect the crystalline structure of TiO2. Furthermore, no diffraction peak corresponding to C70 was observed for the XRD pattern of C70-TiO2 hybrid, suggesting that C70 was well-dispersed in the hybrid22. The representative SEM and HRTEM images of C70-TiO2 hybrid are displayed in Supplementary Fig. S2. As shown in Supplementary Fig. S2a, TiO2 nanoparticles were well dispersed and little aggregated. The surface of hybrid was uneven, which can increase the specific surface area and enhance the photocatalytic activity of C70-TiO2 hybrid23. The presence of C70 cannot be identified from the SEM micrograph, which is ascribed to the undetectable size of C70 at the present resolution. The lattice fringe of anatase phase can be clearly observed and its spacing is measured to be ca. 0.35 nm (see Supplementary Fig. S2b), which is attributed to the TiO2 (101) plane24. However, the outer boundary of the sample is distinctly different. The surface of TiO2 nanoparticle is surrounded by a noncrystal coverture layer with a thickness of about 1 nm, which is close to the diameter of C70 molecule. Thus, the outer layer can be assumed to be C70 which is dispersed on the surface of TiO2 with a monolayer structure25. The UV-Vis diffuse reflectance spectra of C70, TiO2 and C70-TiO2 hybrids are displayed in Supplementary Fig. S3. C70 has strong absorption in the ultraviolet region and weak but significant bands in the visible region. The band gap of TiO2 is estimated to be 3.17 eV. With the doping of C70 into TiO2 materials, hybrids exhibited remarkably enhanced light-harvesting performance in the visible light region, indicating that the C70-TiO2 hybrid can be excited to generate more hole–electron pairs under visible light irradiation due to the C70-sensitized activation.
Comparison of Photocatalytic Disinfection Performance between TiO2 and C70-TiO2 Hybrid. E. coli O157:H7, a notorious waterborne pathogenic microorganism involved in numerous outbreak events causing foodborne and waterborne diseases, was used to investigate the photocatalytic disinfection performance of C70-TiO2 hybrid under visible light irradiation. Figure 1 shows the photocatalytic disinfection efficiencies of E. coli O157:H7 by C70, TiO2 and C70-TiO2 hybrid with the same weight under visible light irradiation (λ ≥ 420 nm). As a comparison, the dark control was carried out with or without the three photocatalysts in the dark. E. coli O157:H7 with or without TiO2 (or C70) in the dark remained mostly viable over a period of 120 min, indicating that TiO2 (or C70) has no bactericidal effect without light irradiation. However, the survival rate of E. coli O157:H7 in the presence of C70-TiO2 in the dark reduced to 70% after 120 min, indicating that C70-TiO2 hybrid has physically toxic effects on E. coli O157:H7. The light control was also performed with or without the three photocatalysts under visible light irradiation. As shown in Fig. 1, only visible light irradiation without any of the three photocatalysts could not disinfect E. coli O157:H7. C70 and TiO2 have moderate toxic effects on E. coli Scientific Reports | 6:25702 | DOI: 10.1038/srep25702
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Figure 2. Effect of the catalyst concentration on the disinfection rate of E. coli O157:H7.
O157:H7 with a disinfection rate of 24% and 30% in 120 min under visible light irradiation, respectively, whereas C70-TiO2 hybrid kills about 73% of E. coli O157:H7 in 120 min, which is significantly higher than that of C70 and TiO2 under visible light irradiation. The process of disinfection can be fitted with Weibull deactivation function and expressed by equation (1) 26: Nt /N0 = e−kt
(1)
where Nt/N0 represents the number of viable cell divided by the total cell count at time t (min), and k is the disinfection rate constant. Under visible light irradiation, the inactivation rate constant k of C70-TiO2 hybrid was 2.3-fold that of TiO2. One possible reason for the higher inactivation efficiency of C70-TiO2 hybrid is that carbon dots, such as fullerenes (C60 and C70) and carbon nanotubes, have strong adsorption ability in the visible light region and can increase the visible light adsorption efficiency of C70-TiO2 hybrid13. As mentioned in the result of DRS, C70-TiO2 composites showed more intensive absorption over the whole visible light region than pure TiO2 (Supplementary Fig. S3). Additionally, C70 can act as electron traps because of its high electron affinities17, which would function as charge separator and enhance the photocatalytic disinfection activity of C70-TiO2 hybrid.
Effect of C70-TiO2 Hybrid Concentration on Photocatalytic Disinfection Performance. The dis-
infection rate of E. coli O157:H7 as a function of C70-TiO2 hybrid concentrations is shown in Fig. 2. With the catalyst dosage increased from 0.25 to 1.00 mg mL−1, the deactivation rate of E. coli O157:H7 increased, followed by a decrease with the dosage further increased to 1.5 mg mL−1. The k values of bacteria were 0.005, 0.007, 0.010 and 0.009 min−1 corresponding to the catalyst dosages of 0.25, 0.50, 1.00, and 1.50 mg/mL, respectively. These results revealed that the optimum catalyst dosage is 1.0 g L−1, suggesting that, with a low dosage, the incident light cannot be completely used by the catalyst, leading to the generation of less active substances. With an increase of the catalyst dosage, more incident light can be absorbed to produce more active substances, which simultaneously can also cause a steady increase of the solution turbidity, resulting in a decline in the utilization of the photons27, and finally the reduction of sterilization efficiency. To further confirm the photocatalytic disinfection performance of C70-TiO2 hybrid, the fluorescence assays were performed by treating E. coli O157:H7 with C70-TiO2 hybrid without or under visible light irradiation (Fig. 3). The control experiment revealed that C70-TiO2 hybrid has no color fluorescence (Fig. 3a) and cannot obstruct the observation on bacteria. The bacteria treated only with C70-TiO2 hybrid can also be observed to exhibit intense green fluorescence (Fig. 3b). In the presence of C70-TiO2 hybrid, after 1 h irradiation by visible light, nearly half of the cells showed red fluorescence (Fig. 3c), and after 2 h irradiation, almost no living E. coli O157:H7 can be observed (Fig. 3d). Since the Live/Dead BacLight stain only shows the integrity of the cell walls of E. coli O157:H728, which cannot demonstrate the culturability of E. coli O157:H7. So we used plate count to investigate the survival rate of E. coli O157:H7 in the photocatalytic disinfection process. The results demonstrate that the cell culturability of E. coli O157:H7 decreased with the extension of irradiation time and was significantly lower than the result measured by the corresponding fluorescent count (see Supplementary Fig. S4). Actually, plate count is not a suitable method to determine the survival rate of the bacteria mixed with other materials. In the process of photocatalytic disinfection, the bacterial cell walls were damaged first, suggesting that the fluorescent staining technique is a direct and accurate method, and thus it was used in the following experiment.
Photocatalytic Disinfection Mechanism. It is well accepted that valence band (VB) holes and conduction band (CB) electrons can be generated when the photocatalyst is irradiated by incident light with a proper wavelength29. VB holes can oxidize the H2O to produce •O H, and the photogenerated CB electrons can reduce the surface absorbed O2 to produce the superoxide radical anion O2−•. The produced •OH and O2−•radicals as well as VB holes can disinfect the bacteria. However, there is still a heated argument about which reactive species plays a more significant role in the photocatalytic disinfection process30,31. Therefore, to address this issue, we used Scientific Reports | 6:25702 | DOI: 10.1038/srep25702
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Figure 3. Fluorescence microscopic images of E. coli O157:H7 (0.5 mg mL−1, 30 mL) treated with C70-TiO2 hybrid (1 mg mL−1) without and under visible light irradiation. (A) Only C70-TiO2 hybrid, (B) the mixture of photocatalyst and E. coli O157:H7 before irradiation, and after irradiation by visible light for (C) 1 h, and (D) 2 h.
Figure 4. Photocatalytic disinfection of E. coli O157:H7 using C70-TiO2 hybrid with different scavengers (0.3 mol L−1 isopropanol, 3.0 × 10−5 mol L−1 sodium oxalate) under visible light irradiation.
different scavengers individually to remove the specific reactive species. Isopropanol and sodium oxalate were used as the scavengers for •OH and VB holes, respectively. It can be seen from Fig. 4 that the isopropanol and sodium oxalate cannot disinfect the E. coli O157:H7 in the absence of photocatalyst. In the presence of sodium oxalate, the photocatalytic disinfection effect of C70-TiO2 is almost the same as that without the scavenger, indicating that VB holes have almost no action in the disinfection. Interestingly, with the addition of isopropanol to remove •OH in bulk, the photocatalytic disinfection effect of C70-TiO2 is evidently inhibited (the survival rate of E. coli O157:H7 rose from 25% to 74% after 2 h irradiation), indicating that •OH generated by the photocatalyst plays an important role in the disinfection of E. coli O157:H7. It is reported that isopropanol can only capture the diffusing •OH in bulk due to its low affinity to the surface of semiconductors in aqueous media32. To further investigate the role of diffusing •OH in the photocatalytic disinfection, we separated E. coli O157:H7 and C70-TiO2 hybrid by using a semipermeable membrane, which only allows water and diffusing •OH to pass through. A suspension of E. coli O157:H7 was contained in the container of semipermeable membrane and C70-TiO2 hybrid was dispersed outside of the container. As shown in Fig. 5, the Scientific Reports | 6:25702 | DOI: 10.1038/srep25702
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Figure 5. Photocatalytic disinfection of E. coli O157:H7 (0.5 mg mL−1) with or without a semipermeable membrane in the suspension of 1 mg mL−1 C70-TiO2 hybrid.
Figure 6. Photocatalytic disinfection of E. coli O157:H7 using C70-TiO2 hybrid as a function of cells with full EPS or partially extracted EPS.
survival rate of E. coli O157:H7 in the semipermeable membrane increased from 25% to 60% after 2 h visible-light irradiation, indicating that the photocatalytic disinfection efficiency of E. coli O157:H7 in the separated system (Fig. 5) is much lower than that in the nonseparated system. This phenomenon indicates that the diffusing •OH is quite important to the disinfection of E. coli O157:H7, which is consistent with the result of Kubacka’s study. Kubacka et al. recently reported a detailed genetic investigation into the mechanism of TiO2 photocatalysis and concluded that extensive radical induced cell wall modifications are the main factor responsible for the high biocidal performance of TiO2-based nanomaterials33. In addition, the above result of our study also suggests that the direct contact between C70-TiO2 hybrid and E. coli O157:H7 is not indispensable for the photocatalytic disinfection. As demonstrated in Zhang’s research34, the destruction of the outer membrane of E. coli K-12 was the initial step in the photocatalytic disinfection. To verify the role of bacterial extracellular polymeric substance (EPS) — an important reactive component associated with bacterial cell walls in the photocatalytic disinfection, we removed EPS from E. coli O157:H7 by using cationic exchange resin (CER)35 and tested the survival rate of EPS-free E. coli O157:H7 in the photocatalytic disinfection. As shown in Fig. 6, there is almost no difference between the disinfection rates of E. coli O157:H7 and EPS-free E. coli O157:H7 either under visible light irradiation or in the dark. As mentioned above, the attack by •O H contributes a large part to the disinfection of E. coli O157:H7. As an electrophilic species, •OH usually does not attack EPS which mainly contains a high molecular weight polymer such as polysaccharides, proteins, and nucleic acids. Only if the amount of •OH is increased to a certain level can the unselective attack on both EPS and cell wall of E. coli O157:H7 by •OH be observed. In real applications of photocatalytic disinfection, the photocatalysts are usually immobilized to facilitate the recovery. Therefore, the photocatalytic disinfection efficiency of C70-TiO2 thin film was examined. The C70-TiO2 thin films were prepared by dispersion, coating and natural drying. The inhibitory effect of C70-TiO2 thin films on E. coli O157:H7 was investigated. Figure 7 shows the growth status of E. coli O157:H7 under different conditions. In the absence of photocatalyst, LB medium in the orifice plate becomes very turbid whether or not in the dark, resulting from the massive growth of bacteria. Furthermore, E. coli O157:H7 retains its viability in the solution containing TiO2 thin film or C70-TiO2 thin film without visible light irradiation. However, the LB medium solution containing visible-light-irradiated C70-TiO2 thin film in the orifice plate is as limpid as before the treatment. Scientific Reports | 6:25702 | DOI: 10.1038/srep25702
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Figure 7. Inhibitory effects of TiO2 and C70-TiO2 thin films on the formation of E. coli O157:H7 biofilm in the dark and under visible light irradiation. Where the square piece is TiO2 or C70-TiO2 thin film, CK refers to control experiment without TiO2 and C70-TiO2 thin film.
The concentration of bacteria dropped from 3 × 108 to 38 cfu/mL in the solution with C70-TiO2 thin film after 2 h irradiation and the growth of E. coli O157:H7 was completely inhibited in the later 24 h irradiation. These experimental results manifested the efficient and durable bactericidal activity of C70-TiO2 thin film under visible light irradiation. To evaluate the reusability of C70-TiO2 thin film, the used thin film was collected from the LB medium after the disinfection experiment, washed with distilled water, and dried for the next cycle. It is noteworthy that after four cycle tests for a total of 100 hours, the C70-TiO2 thin film showed no obvious loss of photocatalytic disinfection activity after the 100 h cycled test (see Supplementary Fig. S5), indicating that C70-TiO2 hybrid is a highly stable photocatalyst for the disinfection of E. coli O157:H7 under visible light irradiation. The overall results suggest that C70-TiO2 hybrid is stable and reusable as a promising efficient photocatalyst for inactivation of waterborne pathogenic microorganisms.
Materials and Methods
Preparation of C70-TiO2 Hybrid. C70 (purity 420 nm). The light intensity was fixed at 600 μW cm−2. After the photocatalytic disinfection treatment, an aliquot of the reaction solution was sampled. The samples were fluorescently stained with the LIVE/DEAD BacLight bacterial viability kit (L13152, Molecular Probes, Inc., Eugene, OR). After incubation in the dark for 15 min, the samples were examined using a fluorescence microscope (Zeiss Imager A1, Germany). The living cells showed green fluorescence under emitted light 460–500 nm and the dead cells showed red fluorescence under emitted light 510–560 nm. The survival ratio of the E. coli O157:H7 was estimated by the number of viable cells divided by the total cell count. As a comparison, a dark control (without light irradiation and photocatalyst), a light control (light irradiation alone without the photocatalyst), and a negative control (without the photocatalyst or light irradiation) were also conducted. All the photocatalytic bacterial disinfection experiments were performed in triplicate. To study whether direct contact between photocatalyst and E. coli O157:H7 is important for disinfection, a partition system37 was used to separate E. coli O157:H7 from the surface of photocatalyst. E. coli O157:H7 suspension was pipetted into a semipermeable container (semipermeable membrane), and the photocatalyst suspension was maintained outside of the membrane and stirred continuously to keep the photocatalyst evenly distributed in the solution. At different intervals, aliquots of the cells inside the membrane were sampled, and the number of viable cells in the samples was determined as described above. The scavenger experiments were carried out by adding individual scavenger to the photocatalytic reaction system. Isopropanol and sodium oxalate were used as the scavengers for •OH and VB holes, respectively10.
References
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (41522106), the National Basic Research Program of China (2015CB150504) and the Doctor Foundation of Shandong (BS2013HZ009).
Author Contributions
P.C. designed the experiments. K.O. performed the experiments. K.O. and K.D. wrote the paper. S.L.W., Q.H. and X.Y. reviewed the manuscript. All authors reviewed the paper.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. How to cite this article: Ouyang, K. et al. Efficient Photocatalytic Disinfection of Escherichia coli O157:H7 using C70-TiO2 Hybrid under Visible Light Irradiation. Sci. Rep. 6, 25702; doi: 10.1038/srep25702 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
Scientific Reports | 6:25702 | DOI: 10.1038/srep25702
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received: 11 December 2015 accepted: 21 April 2016 Published: 10 May 2016
Efficient Photocatalytic Disinfection of Escherichia coli O157:H7 using C70-TiO2 Hybrid under Visible Light Irradiation Kai Ouyang1,*, Ke Dai1,*, Sharon L. Walker2, Qiaoyun Huang1, Xixiang Yin3 & Peng Cai1 Efficient photocatalytic disinfection of Escherichia coli O157:H7 was achieved by using a C70 modified TiO2 (C70-TiO2) hybrid as a photocatalyst under visible light (λ > 420 nm) irradiation. Disinfection experiments showed that 73% of E. coli O157:H7 died within 2 h with a disinfection rate constant of k = 0.01 min−1, which is three times that measured for TiO2. The mechanism of cell death was investigated by using several scavengers combined with a partition system. The results revealed that diffusing hydroxyl radicals play an important role in the photocatalytically initiated bacterial death, and direct contact between C70-TiO2 hybrid and bacteria is not indispensable in the photocatalytic disinfection process. Extracellular polymeric substances (EPS) of bacteria have little effect on the disinfection efficiency. Analyses of the inhibitory effect of C70-TiO2 thin films on E. coli O157:H7 showed a decrease of the bacterial concentration from 3 × 108 to 38 cfu mL−1 in the solution with C70-TiO2 thin film in the first 2 h of irradiation and a complete inhibition of the growth of E. coli O157:H7 in the later 24 h irradiation. Millions of people die from diseases transmitted through contaminated water or human excreta every year1. As a waterborne pathogenic microorganism, Escherichia coli O157:H7 reportedly causes an estimated 73,480 illnesses, 2,168 hospitalizations, and 61 deaths each year in the United States2. Reliable, efficient, and environmentally safe disinfection has become an increasing concern throughout the world3. Traditional water disinfection methods such as chemical oxidation technologies can require expensive chemicals or costly equipment. Moreover, unwanted oxidation bi-products may be formed in water from chlorination or ozonation processes4. Although UV radiation is capable of sustained disinfection, the hazards of intensive and direct use of UV radiation limit its application5. Therefore, the development of an efficient and environmental friendly disinfection technology has become an urgent demand. Since Matsunaga et al. discovered the bactericidal activity of TiO2 as a photocatalyst for the first time in 19856, many studies related to the bactericidal effect of TiO2 photocatalyst have been reported, including the death of bacteria and inactivation of viruses7,8. However, the potential for the practical application of TiO2 is restricted by the broad band gap (3.2 eV) of anatase TiO2 and low quantum efficiency9. Recently, considerable effort has been taken to address these issues by doping the TiO2 with non-metal or metal elements or by coupling with narrow band semiconductors10–12. Carbon nanomaterials (fullerene, carbon nanotube, and graphene) are highly valued because of their excellent optical performance, small size effect, and ease in achieving surface functionalization towards coupling with TiO213. C60 has drawn particular attention of researchers due to its being an excellent electron acceptor with delocalized conjugated structure14–16, which can efficiently separate photo-induced charge. C70 is a close-shell configuration consisting of 35 bonding molecular orbitals with 70 p-electrons, which can promote efficient electron transfer reduction17. Compared with C60, C70 has higher electron affinity and higher possibility to form anions or free radicals because of the reduced symmetry structure18. In addition, C70 has a larger photo cross-sectional area, which suggests that a high light harvesting efficiency can be achieved19. Latest 1 State Key Laboratory of Agricultural Microbiology, College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China. 2Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521, USA. 3Jinan Research Academy of Environmental Sciences, Jinan 250014, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to P.C. (email: [email protected])
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Figure 1. Survival of E. coli O157:H7 (0.5 mg mL−1) presented as the ratio of live to total cells (Nt/N0) versus the time. Suspensions of cells were exposed to dispersions containing different catalysts (1 mg mL−1) in the dark and under visible light irradiation.
studies revealed that C70-TiO2 hybrids show a significant photocatalytic activity for degradation of organic pollutant in visible light region20,21. The aforementioned reports imply that a high efficient bactericidal activity can be expected from the C70-TiO2 hybrid. In the present study, we prepared a C70-TiO2 hybrid by a hydrothermal reaction process and investigated this material’s capacity for photocatalytic disinfection of E. coli O157:H7 under visible light irradiation. The effects of the photocatalyst dosages and the contact modes between C70-TiO2 hybrid and bacterial cells on the photocatalytic disinfection rates were investigated. Several scavengers for hydroxyl radicals and valence-band holes were applied to examine the roles of the oxidative species in the photocatalytic disinfection of E. coli O157:H7. Moreover, the effect of bacterial extracellular polymeric substance (EPS) on the photocatalytic disinfection was also evaluated.
Results and Discussion
Crystal Phase Composition, Morphology and Optical Properties of C70-TiO2 Hybrid. The XRD
patterns of TiO2 and C70-TiO2 hybrid are shown in Supplementary Fig. S1. The peaks at 25.4° (101), 37.5° (004), 48.1° (200), 53.8° (105), 54.8° (211), and 62.8° (204) corresponded to the pure anatase phase. It can also be seen that the diffraction peaks of anatase phase in C70-TiO2 hybrid did not shift with the incorporation of C70, indicating that the introduction of C70 does not affect the crystalline structure of TiO2. Furthermore, no diffraction peak corresponding to C70 was observed for the XRD pattern of C70-TiO2 hybrid, suggesting that C70 was well-dispersed in the hybrid22. The representative SEM and HRTEM images of C70-TiO2 hybrid are displayed in Supplementary Fig. S2. As shown in Supplementary Fig. S2a, TiO2 nanoparticles were well dispersed and little aggregated. The surface of hybrid was uneven, which can increase the specific surface area and enhance the photocatalytic activity of C70-TiO2 hybrid23. The presence of C70 cannot be identified from the SEM micrograph, which is ascribed to the undetectable size of C70 at the present resolution. The lattice fringe of anatase phase can be clearly observed and its spacing is measured to be ca. 0.35 nm (see Supplementary Fig. S2b), which is attributed to the TiO2 (101) plane24. However, the outer boundary of the sample is distinctly different. The surface of TiO2 nanoparticle is surrounded by a noncrystal coverture layer with a thickness of about 1 nm, which is close to the diameter of C70 molecule. Thus, the outer layer can be assumed to be C70 which is dispersed on the surface of TiO2 with a monolayer structure25. The UV-Vis diffuse reflectance spectra of C70, TiO2 and C70-TiO2 hybrids are displayed in Supplementary Fig. S3. C70 has strong absorption in the ultraviolet region and weak but significant bands in the visible region. The band gap of TiO2 is estimated to be 3.17 eV. With the doping of C70 into TiO2 materials, hybrids exhibited remarkably enhanced light-harvesting performance in the visible light region, indicating that the C70-TiO2 hybrid can be excited to generate more hole–electron pairs under visible light irradiation due to the C70-sensitized activation.
Comparison of Photocatalytic Disinfection Performance between TiO2 and C70-TiO2 Hybrid. E. coli O157:H7, a notorious waterborne pathogenic microorganism involved in numerous outbreak events causing foodborne and waterborne diseases, was used to investigate the photocatalytic disinfection performance of C70-TiO2 hybrid under visible light irradiation. Figure 1 shows the photocatalytic disinfection efficiencies of E. coli O157:H7 by C70, TiO2 and C70-TiO2 hybrid with the same weight under visible light irradiation (λ ≥ 420 nm). As a comparison, the dark control was carried out with or without the three photocatalysts in the dark. E. coli O157:H7 with or without TiO2 (or C70) in the dark remained mostly viable over a period of 120 min, indicating that TiO2 (or C70) has no bactericidal effect without light irradiation. However, the survival rate of E. coli O157:H7 in the presence of C70-TiO2 in the dark reduced to 70% after 120 min, indicating that C70-TiO2 hybrid has physically toxic effects on E. coli O157:H7. The light control was also performed with or without the three photocatalysts under visible light irradiation. As shown in Fig. 1, only visible light irradiation without any of the three photocatalysts could not disinfect E. coli O157:H7. C70 and TiO2 have moderate toxic effects on E. coli Scientific Reports | 6:25702 | DOI: 10.1038/srep25702
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Figure 2. Effect of the catalyst concentration on the disinfection rate of E. coli O157:H7.
O157:H7 with a disinfection rate of 24% and 30% in 120 min under visible light irradiation, respectively, whereas C70-TiO2 hybrid kills about 73% of E. coli O157:H7 in 120 min, which is significantly higher than that of C70 and TiO2 under visible light irradiation. The process of disinfection can be fitted with Weibull deactivation function and expressed by equation (1) 26: Nt /N0 = e−kt
(1)
where Nt/N0 represents the number of viable cell divided by the total cell count at time t (min), and k is the disinfection rate constant. Under visible light irradiation, the inactivation rate constant k of C70-TiO2 hybrid was 2.3-fold that of TiO2. One possible reason for the higher inactivation efficiency of C70-TiO2 hybrid is that carbon dots, such as fullerenes (C60 and C70) and carbon nanotubes, have strong adsorption ability in the visible light region and can increase the visible light adsorption efficiency of C70-TiO2 hybrid13. As mentioned in the result of DRS, C70-TiO2 composites showed more intensive absorption over the whole visible light region than pure TiO2 (Supplementary Fig. S3). Additionally, C70 can act as electron traps because of its high electron affinities17, which would function as charge separator and enhance the photocatalytic disinfection activity of C70-TiO2 hybrid.
Effect of C70-TiO2 Hybrid Concentration on Photocatalytic Disinfection Performance. The dis-
infection rate of E. coli O157:H7 as a function of C70-TiO2 hybrid concentrations is shown in Fig. 2. With the catalyst dosage increased from 0.25 to 1.00 mg mL−1, the deactivation rate of E. coli O157:H7 increased, followed by a decrease with the dosage further increased to 1.5 mg mL−1. The k values of bacteria were 0.005, 0.007, 0.010 and 0.009 min−1 corresponding to the catalyst dosages of 0.25, 0.50, 1.00, and 1.50 mg/mL, respectively. These results revealed that the optimum catalyst dosage is 1.0 g L−1, suggesting that, with a low dosage, the incident light cannot be completely used by the catalyst, leading to the generation of less active substances. With an increase of the catalyst dosage, more incident light can be absorbed to produce more active substances, which simultaneously can also cause a steady increase of the solution turbidity, resulting in a decline in the utilization of the photons27, and finally the reduction of sterilization efficiency. To further confirm the photocatalytic disinfection performance of C70-TiO2 hybrid, the fluorescence assays were performed by treating E. coli O157:H7 with C70-TiO2 hybrid without or under visible light irradiation (Fig. 3). The control experiment revealed that C70-TiO2 hybrid has no color fluorescence (Fig. 3a) and cannot obstruct the observation on bacteria. The bacteria treated only with C70-TiO2 hybrid can also be observed to exhibit intense green fluorescence (Fig. 3b). In the presence of C70-TiO2 hybrid, after 1 h irradiation by visible light, nearly half of the cells showed red fluorescence (Fig. 3c), and after 2 h irradiation, almost no living E. coli O157:H7 can be observed (Fig. 3d). Since the Live/Dead BacLight stain only shows the integrity of the cell walls of E. coli O157:H728, which cannot demonstrate the culturability of E. coli O157:H7. So we used plate count to investigate the survival rate of E. coli O157:H7 in the photocatalytic disinfection process. The results demonstrate that the cell culturability of E. coli O157:H7 decreased with the extension of irradiation time and was significantly lower than the result measured by the corresponding fluorescent count (see Supplementary Fig. S4). Actually, plate count is not a suitable method to determine the survival rate of the bacteria mixed with other materials. In the process of photocatalytic disinfection, the bacterial cell walls were damaged first, suggesting that the fluorescent staining technique is a direct and accurate method, and thus it was used in the following experiment.
Photocatalytic Disinfection Mechanism. It is well accepted that valence band (VB) holes and conduction band (CB) electrons can be generated when the photocatalyst is irradiated by incident light with a proper wavelength29. VB holes can oxidize the H2O to produce •O H, and the photogenerated CB electrons can reduce the surface absorbed O2 to produce the superoxide radical anion O2−•. The produced •OH and O2−•radicals as well as VB holes can disinfect the bacteria. However, there is still a heated argument about which reactive species plays a more significant role in the photocatalytic disinfection process30,31. Therefore, to address this issue, we used Scientific Reports | 6:25702 | DOI: 10.1038/srep25702
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Figure 3. Fluorescence microscopic images of E. coli O157:H7 (0.5 mg mL−1, 30 mL) treated with C70-TiO2 hybrid (1 mg mL−1) without and under visible light irradiation. (A) Only C70-TiO2 hybrid, (B) the mixture of photocatalyst and E. coli O157:H7 before irradiation, and after irradiation by visible light for (C) 1 h, and (D) 2 h.
Figure 4. Photocatalytic disinfection of E. coli O157:H7 using C70-TiO2 hybrid with different scavengers (0.3 mol L−1 isopropanol, 3.0 × 10−5 mol L−1 sodium oxalate) under visible light irradiation.
different scavengers individually to remove the specific reactive species. Isopropanol and sodium oxalate were used as the scavengers for •OH and VB holes, respectively. It can be seen from Fig. 4 that the isopropanol and sodium oxalate cannot disinfect the E. coli O157:H7 in the absence of photocatalyst. In the presence of sodium oxalate, the photocatalytic disinfection effect of C70-TiO2 is almost the same as that without the scavenger, indicating that VB holes have almost no action in the disinfection. Interestingly, with the addition of isopropanol to remove •OH in bulk, the photocatalytic disinfection effect of C70-TiO2 is evidently inhibited (the survival rate of E. coli O157:H7 rose from 25% to 74% after 2 h irradiation), indicating that •OH generated by the photocatalyst plays an important role in the disinfection of E. coli O157:H7. It is reported that isopropanol can only capture the diffusing •OH in bulk due to its low affinity to the surface of semiconductors in aqueous media32. To further investigate the role of diffusing •OH in the photocatalytic disinfection, we separated E. coli O157:H7 and C70-TiO2 hybrid by using a semipermeable membrane, which only allows water and diffusing •OH to pass through. A suspension of E. coli O157:H7 was contained in the container of semipermeable membrane and C70-TiO2 hybrid was dispersed outside of the container. As shown in Fig. 5, the Scientific Reports | 6:25702 | DOI: 10.1038/srep25702
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Figure 5. Photocatalytic disinfection of E. coli O157:H7 (0.5 mg mL−1) with or without a semipermeable membrane in the suspension of 1 mg mL−1 C70-TiO2 hybrid.
Figure 6. Photocatalytic disinfection of E. coli O157:H7 using C70-TiO2 hybrid as a function of cells with full EPS or partially extracted EPS.
survival rate of E. coli O157:H7 in the semipermeable membrane increased from 25% to 60% after 2 h visible-light irradiation, indicating that the photocatalytic disinfection efficiency of E. coli O157:H7 in the separated system (Fig. 5) is much lower than that in the nonseparated system. This phenomenon indicates that the diffusing •OH is quite important to the disinfection of E. coli O157:H7, which is consistent with the result of Kubacka’s study. Kubacka et al. recently reported a detailed genetic investigation into the mechanism of TiO2 photocatalysis and concluded that extensive radical induced cell wall modifications are the main factor responsible for the high biocidal performance of TiO2-based nanomaterials33. In addition, the above result of our study also suggests that the direct contact between C70-TiO2 hybrid and E. coli O157:H7 is not indispensable for the photocatalytic disinfection. As demonstrated in Zhang’s research34, the destruction of the outer membrane of E. coli K-12 was the initial step in the photocatalytic disinfection. To verify the role of bacterial extracellular polymeric substance (EPS) — an important reactive component associated with bacterial cell walls in the photocatalytic disinfection, we removed EPS from E. coli O157:H7 by using cationic exchange resin (CER)35 and tested the survival rate of EPS-free E. coli O157:H7 in the photocatalytic disinfection. As shown in Fig. 6, there is almost no difference between the disinfection rates of E. coli O157:H7 and EPS-free E. coli O157:H7 either under visible light irradiation or in the dark. As mentioned above, the attack by •O H contributes a large part to the disinfection of E. coli O157:H7. As an electrophilic species, •OH usually does not attack EPS which mainly contains a high molecular weight polymer such as polysaccharides, proteins, and nucleic acids. Only if the amount of •OH is increased to a certain level can the unselective attack on both EPS and cell wall of E. coli O157:H7 by •OH be observed. In real applications of photocatalytic disinfection, the photocatalysts are usually immobilized to facilitate the recovery. Therefore, the photocatalytic disinfection efficiency of C70-TiO2 thin film was examined. The C70-TiO2 thin films were prepared by dispersion, coating and natural drying. The inhibitory effect of C70-TiO2 thin films on E. coli O157:H7 was investigated. Figure 7 shows the growth status of E. coli O157:H7 under different conditions. In the absence of photocatalyst, LB medium in the orifice plate becomes very turbid whether or not in the dark, resulting from the massive growth of bacteria. Furthermore, E. coli O157:H7 retains its viability in the solution containing TiO2 thin film or C70-TiO2 thin film without visible light irradiation. However, the LB medium solution containing visible-light-irradiated C70-TiO2 thin film in the orifice plate is as limpid as before the treatment. Scientific Reports | 6:25702 | DOI: 10.1038/srep25702
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Figure 7. Inhibitory effects of TiO2 and C70-TiO2 thin films on the formation of E. coli O157:H7 biofilm in the dark and under visible light irradiation. Where the square piece is TiO2 or C70-TiO2 thin film, CK refers to control experiment without TiO2 and C70-TiO2 thin film.
The concentration of bacteria dropped from 3 × 108 to 38 cfu/mL in the solution with C70-TiO2 thin film after 2 h irradiation and the growth of E. coli O157:H7 was completely inhibited in the later 24 h irradiation. These experimental results manifested the efficient and durable bactericidal activity of C70-TiO2 thin film under visible light irradiation. To evaluate the reusability of C70-TiO2 thin film, the used thin film was collected from the LB medium after the disinfection experiment, washed with distilled water, and dried for the next cycle. It is noteworthy that after four cycle tests for a total of 100 hours, the C70-TiO2 thin film showed no obvious loss of photocatalytic disinfection activity after the 100 h cycled test (see Supplementary Fig. S5), indicating that C70-TiO2 hybrid is a highly stable photocatalyst for the disinfection of E. coli O157:H7 under visible light irradiation. The overall results suggest that C70-TiO2 hybrid is stable and reusable as a promising efficient photocatalyst for inactivation of waterborne pathogenic microorganisms.
Materials and Methods
Preparation of C70-TiO2 Hybrid. C70 (purity 420 nm). The light intensity was fixed at 600 μW cm−2. After the photocatalytic disinfection treatment, an aliquot of the reaction solution was sampled. The samples were fluorescently stained with the LIVE/DEAD BacLight bacterial viability kit (L13152, Molecular Probes, Inc., Eugene, OR). After incubation in the dark for 15 min, the samples were examined using a fluorescence microscope (Zeiss Imager A1, Germany). The living cells showed green fluorescence under emitted light 460–500 nm and the dead cells showed red fluorescence under emitted light 510–560 nm. The survival ratio of the E. coli O157:H7 was estimated by the number of viable cells divided by the total cell count. As a comparison, a dark control (without light irradiation and photocatalyst), a light control (light irradiation alone without the photocatalyst), and a negative control (without the photocatalyst or light irradiation) were also conducted. All the photocatalytic bacterial disinfection experiments were performed in triplicate. To study whether direct contact between photocatalyst and E. coli O157:H7 is important for disinfection, a partition system37 was used to separate E. coli O157:H7 from the surface of photocatalyst. E. coli O157:H7 suspension was pipetted into a semipermeable container (semipermeable membrane), and the photocatalyst suspension was maintained outside of the membrane and stirred continuously to keep the photocatalyst evenly distributed in the solution. At different intervals, aliquots of the cells inside the membrane were sampled, and the number of viable cells in the samples was determined as described above. The scavenger experiments were carried out by adding individual scavenger to the photocatalytic reaction system. Isopropanol and sodium oxalate were used as the scavengers for •OH and VB holes, respectively10.
References
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (41522106), the National Basic Research Program of China (2015CB150504) and the Doctor Foundation of Shandong (BS2013HZ009).
Author Contributions
P.C. designed the experiments. K.O. performed the experiments. K.O. and K.D. wrote the paper. S.L.W., Q.H. and X.Y. reviewed the manuscript. All authors reviewed the paper.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. How to cite this article: Ouyang, K. et al. Efficient Photocatalytic Disinfection of Escherichia coli O157:H7 using C70-TiO2 Hybrid under Visible Light Irradiation. Sci. Rep. 6, 25702; doi: 10.1038/srep25702 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
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